The objective of this research is to gain a more detailed understanding of the relationship between the structure of RNA and ribosomes and their function in protein synthesis. To this end the role of 5s RNA in the structure and function of the 50s ribosomal subunit will be investigated. A procedure has been developed which results in the reversible release of 5s RNA, together with several ribosomal proteins, from bacterial ribosomes. The reconstitution process will be studied by examining in detail the binding of 5s RNA and modified 5s RNA to the ribosome and the requirement of release proteins, individually and in various combinations, for this binding. Physical-chemical characterization of completely and partially reconstituted ribosomes and the activity of these ribosomes in polypeptide formation will provide information on the localization of 5s RNA within the 50s subunit and on its function protein biosynthesis. A second approach to the objective involves investigating the molecular mechanisms of the effects produced by the pyrimidine analogue 5-fluorouracil (FU) on the structure, function and synthesis of RNA. Our previous studies had shown that FU is incorporated into bacterial RNA and leads to the formation of abnormal ribosomal particles (FU-particles). To learn more about the function of ribosomal RNA (rRNA) and its role in ribosome structure, these altered ribosomes and their RNA and protein constituents will be characterized. FU-containing rRNA will be used in attempts to reconsitute ribosomes from isolated RNA and ribosomal proteins; the properties of the resulting nucleoprotein particles will be examined. A detailed investigation of the effects of FU on bacterial ribosomal RNA synthesis may shed light on the nature of the primary transcription products of the rRNA genes. Incorporation of FU into tRNA alters its structure and properties. Among other things the level of several minor nucleotide components is reduced. The physical-chemical properties of these modified tRNAs, both mixtures and individual species, and their ability to function in accepting amino acids and transferring them into polypeptides will be examined.